Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri) Hyeon-Kyeong JO*ᆞJun-Young CHAE*ᆞHyung-Ho LEE
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(2) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE necessary to study their spawning ecology, and it is. sequence (Schreiber et al., 2001). These regions. thought that molecular biological research data on. were generally ignored in early studies exploring. inshore hagfish must be evaluated.. the structural determinants of PKC isoform function. Protein kinase C (PKC) is a family of enzymes. (Steinberg, 2008). However, V5 domains have. involved in a wide range of biological functions.. recently emerged as structures that impart important. PKCs. been. determinants of PKC isoform-specific targeting and. categorized into conventional (α, βI, βII and. function, suggesting that V5 domains may represent. γ), novel (δ, ε, θ and η) and atypical (ζ. novel. and ι/λ) classes (Newton, 1995). The regulatory. regulate PKC isoform-specific signaling in cells. domains of conventional PKC (cPKC) isoforms. (Bobeszko et al., 2004; Cole and Igumenova, 2015;. contain. Newton, 1995).. have. a. 11. C1. subtypes,. domain. which. that. have. functions. as. a. DAG-/PMA-binding motif (Baneyx and Mujacic,. targets. PKCβI. for. and. pharmaceuticals. PKCβII. are. designed. expressed. in. to. a. 2004). cPKC regulatory domains also contain a C2. tissue-specific and developmentally regulated manner. domain that binds anionic phospholipids in a. (Gopal and Kumar, 2013). RACK1 anchors PKCβ. calcium-dependent manner (Blakey et al., 2005).. II (but not PKCβI) to the perinuclear region; a. Novel PKCs (nPKCs) also have twin C1 domains. PKCβI selective RACK protein is yet to be. as well as a C2 domain. Importantly, nPKC C2. identified. domains lack the critical calcium-coordinating acidic. binding sites were initially mapped to the PKCβ. residues.. Atypical. al.,. 1989).. While. RACK1. lack. a. C2 domain, C2 domain RACK1 binding sites. an. (which are common to PKCβI and PKCβII) do. atypical C1 domain that binds PIP3 or ceramide. not explain the in vivo specificity of RACK1 for. instead.. C2. PKCβI. (aPKCs). et. contain. calcium-sensitive. PKCs. (Ono. domain; and. they. βII. activated. by. PKCβII. (Ohno. et. al.,. 1968).. Rather,. the. diacylglycerol (DAG) and calcium ions perform a. RACK1-binding specificity has been attributed to. wide. regarding. protein-protein interaction motifs in the V5 domain. proliferation, apoptosis, differentiation, motility, and. range. of. that are unique to PKCβII (₆₂₁ACGRNAE⁶²⁷, ₆. inflammation. ₄₅QEVIRN⁶⁵⁰,. with. biological cellular. functions signal. transduction. and. ₆₆₀SFVNSEFLKPEVKS⁶⁷³). (Kawakami et al., 2002; Newton, 1995; Wightman. and not found in PKCβI (Donoghue and Purnell,. and Raetz, 1984).. 2005).. PKCβI and βII have four identical conserved. Thus, we carried out the molecular research on. (C1-C4) regions, but they have about 50 different. PKCβI with wide physiology function in inshore. amino acids in the C-terminal called V5 domains.. hagfish.. V5 domains have 50-70 amino acid sequences; COOH-terminal to the catalytic core of the enzyme. Ⅱ. Materials and Methods. (C3 and C4 domains) that contain the highly conserved turn and hydrophobic phosphorylation motifs as well as an additional 7-21 residues at the extreme COOH terminus (beyond the hydrophobic. 1. cDNA cloning for the complete coding sequence of Eptatretus burgeri PKCβI and βII. motif), that are highly variable in both length and. - 1680 -.
(3) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri) The total RNA was obtained from 11 tissues (brain, tentacle, gill, root of the tongue, spinal cord,. <Table 1> and employed in order to amplify cDNAs from the inshore hagfish cDNA library.. heart, liver, gut, muscle, skin, and mucous gland). 3′ cDNA library screening was conducted with. of inshore hagfish using GeneAll® Hybrid-R™. 3′ GSP (gene specific primer) (sense primer,. Total. Co., Ltd.,. 3’GSP-EbPKCβ-F1 and 3’GSP-EbPKCβ-F2, Table. Korea) as described by previous manuscripts. The. RNA (GeneAll Biotechnology. 1) and Universal primers. In order to isolate the. RNA was reverse-transcribed by oligo (dT)18 using. full-length. the PrimeScript™ 1st strand cDNA Synthesis Kit. conducted using the GeneRacer™ Kit (Invitrogen,. (TaKaRa, Korea) and random hexamer primers.. Korea). 5′ RACE-PCR clone was amplified with. Several PKCβ nucleotide sequences were collected. gene. from. 5’GSP-EbPKCβ-R1. other. species.. Degenerated. primers. were. EbPKCβ,. specific. 5′. primer. RACE-PCR. (antisense. and. was. primer,. 5’GSP-EbPKCβ-R2,. designed around the highly conserved parts of the. <Table 1> and Universal primers. Next, the PCR. sequences using BioEdit Sequence Alignment Editor. product. version 5.0.9 (sense primers, DgPKCβ-F1 and. (GeneAll, Korea). The purified products were then. DgPKCβ-F2; antisense primer, DgPKCβ-R1 and. ligated into pGEM T-Easy vector (Promega, Korea).. DgPKCβ-R2,. Finally, database searches were performed using the. <Table 1> Oligonucleotide primers used in PCR amplification of PKCβI and βII genes of E. burgeri (F, Forward; R, Reverse). was. recovered. by. GeneAll®. SV. gel. BLAST (Basic Local Alignment Search Tool) at the NCBI. (National. Center. for. Biotechnology. Information).. 2. Sequence and phylogenetic analysis Nucleotide and predicted peptide sequences of E. burgeri PKCβI and βII (EbPKCβI and EbPKCβ II) were analyzed using DNASIS for Windows version. 2.5. (Hitachi. software. engineering. Co.,. Japan), BioEdit Sequence Alignment Editor, and BLAST programs in the non-redundant databases of the NCBI (http://www.ncbi.nlm. nih.gov\/BLAST/). Multiple alignments of EbPKCβI and βII amino acid sequences were analyzed using CLUSTAL W version 1.8. The identities and homologies between amino acid sequences were analyzed using BioEdit Sequence. Alignment Editor. version. 5.0.9.. The. multiple sequence alignment obtained was used in order. to. generate. a. phylogenetic. tree. using. neighbor-joining methods, and the reliability of the trees was evaluated using the bootstrap method. - 1681 -.
(4) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE with 1,000 replications. In order to identify possible. were measured by qPCR analysis. The total RNA. phylogenetic clade, a neighbor-joining tree was. extraction and reverse transcription was obtained. generated based on this genetic distance matrix. through the same process described above (2). For. using. the tissue distributions of EbPKCβI and βII, 18s. Kimura. 2-parameter. model. included. in. MEGA 6.. rRNA was used as an internal control gene. The specific. 3. Tissue specific expression patterns of EbPKCβI and βII by qRT-PCR analysis The mRNA distribution of EbPKCβI and βII. primers. Eb18s-rRNA-RT-R, I-RT-R,. Eb18s-rRNA-RT-F,. EbPKCβI-RT-F,. EbPKCβII-RT-F,. and. were used for qPCR(<Table 1>).. - 1682 -. EbPKCβ. EbPKCβII-RT-R.
(5) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri). [Fig. 1] Nucleotide sequence and deduced amino acid sequence of EbPKCβI. Shaded sequences indicate C1, C2, Kinase, and V5 domains. The four circles are lipid cofactor binding surface inside the C1 domain. The two squares are calcium-binding Asp and the square brackets are RACK binding sites. The three triangles indicate ATP binding site (GXGXXG). In the Kinase domain, the first circle is invariant Lys and the second circle is Met as a gatekeeper residue. An underlined Trp is the turn motif and an underlined Ser is the hydrophobic motif. Asterisk (*) at the end of amino acid sequence shows the stop codon. LightCycler® 480 SYBR Green I Master (Roche,. PCR System (Roche) using the following program:. Switzerland) was used in order to monitor the. pre-incubation at 95°C for 5 min, 30 cycles at. quantitative real-time PCR of mRNA transcript. 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s.. abundance on the LightCycler® 480 II Real-Time. The qPCR mixture was made up of the following. - 1683 -.
(6) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE components: 10 µl of 2X SYBR (Roche), 7.5 µl of SYBR water (Roche), 1 µl of sense primer, 1 µl of antisense primer, and 0.5 µl diluted first-strand cDNA (diluted at 1:20) The. ΔΔCT. in. order. In order to construct an expression vector for the suitable production of recombinant EbPKCβI and. method was adopted in order to. calculate the data and the 2−ΔΔCt method was adopted. 4. Expression and purification of recombinant EbPKCβI and βII in E. coli. to. calculate. the. quantitative value (Giulietti et al, 2001).. relative. βII in E. coli, the open reading frame (ORF) of EbPKCβI was amplified by PCR using the primers (sense primer, EcoRI-EbPKCβI-F; and antisense primer XhoI-EbPKCβI-R, as shown in <Table 1>).. - 1684 -.
(7) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri). [Fig. 2] Nucelotide sequence and deduced amino acid sequence of EbPKCβII. Shaded sequences indicate C1, C2, Kinase, and V5 domains. The four circles are lipid cofactor binding surface inside the C1 domain. The two squares are calcium-binding Asp and the square brackets are RACK binding sites. The three triangles indicate ATP binding site (GXGXXG). In the Kinase domain, the first circle is invariant Lys and the second circle is Met as a gatekeeper residue. An underlined Trp is the turn motif and an underlined Ser is the hydrophobic motif. Asterisk(*) at the end of amino acid sequence shows the stop codon. In addition, in the case of EbPKCβII, ORF was. recombinant. plasmids. (EbPKCβI/pET32b. and. amplified by PCR using the primers (sense primer,. EbPKCβII/pET32b) were transformed into E. coli. EcoRI-EbPKCβI-F. primer. strain BL21 (DE3). Transformed cells were grown. XhoI-EbPKCβII-R, as shown in <Table 1>). They. in LB broth (5 ml) containing ampicillin (100. have. the. same. and. the. µg/ml) at 37°C for about 12 h, re-inoculated in. beginning of ORF, so the same sense primer was. two LB broths (5ml) containing ampicillin (100. used in both cases. The amplified fragment was. µg/ml), and grown at 37°C until the OD₆₀₀=0.6.. cloned. Only one of the two LB broths (5 ml) had IPTG. into. the. nucleotide. antisense. pET32b. sequence. at. (Novagen).. The. - 1685 -.
(8) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE. - 1686 -.
(9) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri). - 1687 -.
(10) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE. [Fig. 3] Multiple amino acids sequence alignment analysis of PKC subtypes in various species. The GenBank accession numbers used in the alignment are shown in table 2. Identical amino acid residues are darkly shaded, similar amino acids are lightly shaded, unrelated residues have a white background, and amino acid numbers are shown on the right. “HX₁₂CX₂CX₁₃CX₂CX₄HX ₂C₇C” in the first square box is C1 motif, that exists in both C1A and C1B. The two circles above the sequence show the Asp residues binding to calcium. The second square box shows the ATP binding site, “GXGXXG”. The first inverted triangle is invariant Lys and the second inverted triangle is Met as a gatekeeper residue. (Isopropyl-β-D-thiogalactopyranoside) added to it to. Following electrophoresis, the gels were stained. a final concentration of 1 mM and grown at 20°C. with Coomassie brilliant blue R-250.. about 24 h. The induction of the target proteins. Western blotting was performed using rabbit. was checked by SDS-PAGE (10% running gel, 5%. polyclonal anti-His antibody (1:2000, Santa Cruz. stacking gel) and Western blotting. In order to. Biotechnology). obtain target proteins (EbPKCβI and βII) in large. antibody. scale, the cells were inoculated in LB broth (500. Prestained. mL) and grown at 37°C until the OD₆₀₀=0.6.. USA) were run as standards. The electrophoresed samples. 5. SDS-PAGE and Western blot were denatured in buffer containing 60mM Tris/pH 25%. glycerol,. 2%. SDS,. 14.4mM. 2-mercaptoethanol, and 0.1% bromophenol blue, then boiled for 5min. Purified EbPKCβI and βII were USA).. separated. by. Prestained. 10% SDS-PAGE molecular. weight. molecular were. rabbit Santa. monoclonal Cruz. weight. transferred. markers to. anti-His. Biotechnology). (Bio-Rad,. nitrocellulose. membranes (Schleicher & Schuell. Co., USA) using. For the electrophoresis procedures, all samples 6.8,. and. (1:1000,. (Bio-Rad, markers. (Bio-Rad, USA) were run as standards on each gel.. a Hoefer transblotting system (Pharmacia. Co., USA). Following this transfer, the membrane was blocked with 3% BSA in TPBS [200 mM Tris (pH 7.0), 1.37 M NaCl, 1% Tween-20] for 30 min at room temperature. Primary antibody was attached to the target proteins at 4℃ for 16 h. Secondary antibody was attached to the target proteins at 4℃ for 1.5 h.. - 1688 -.
(11) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri) 6. Activity assay. gland. In order to isolate full-length inshore hagfish. Phosphorylation by PKC of its specific substrate alters the peptide's net charge from +1 to –1. PepTag® Non-Radioactive PKC Assay (Promega) was used to analyze the activities of EbPKCβI and EbPKCβII. For qualitative analysis, proteins were diluted to 1, 2, 5, and 10 ng/μl, and reacted with substrate at 30℃ for 30 min. After reaction, samples underwent electrophoresis on gel, which was made of 50 mM Tris-HCl(pH 8.0) and 0.8% agarose, over 20 min. Phosphorylated peptide was separated for quantitative analysis. At 95℃, it was completely melted and Gel Solubilization Solution, glacial acetic acid, and distilled water were added. Using. a. spectrophotometer,. we. assessed. the. absorbance at A650 and calculated activity using the following equation:. PKCβI and βII, the partial sequences were used as bases for gene-specific primers for RACE PCR. As a result, the full nucleotide sequences of EbPKCβI and EbPKCβII were 2,499 bp and 2,658 bp, respectively. The EbPKCβI sequence was composed of a 238 bp 5’-untranslated region (5’-UTR), a 2,007 bp coding region, and a 254 bp 3’-untranslated region (3’-UTR) [Fig. 1]. 3’-UTR of EbPKCβI had a miR-199-5p binding site from 73 to 78 (5’-TACTGG-3’). The EbPKCβII sequence was composed of a 235 bp 5’-untranslated region (5’-UTR), a 2,004 bp coding region, and a 419 bp 3’-untranslated region (3’-UTR) [Fig. 2]. 3’-UTR of EbPKCβII had a miR-203a-5p binding site from 328 to 334 (5’-GATCCAT-3’). The EbPKCβI codes 668 amino acids, which the molecular weight is approximately 76.43 kDa, and the EbPKCβII. A = εBC,. codes 667 amino acids, which the molecular weight. where: A = absorbance of the sample, ε = the -1. is approximately 76.08 kDa. These sequences were. molar absorptivity of the peptide in L/mol • cm ,. submitted. B = the width of the light cell, and C = the. I(MH350863), PKCβII(MH350864)].. concentration of the peptide in mol/L of the sample. to. EbPKCβI. the and. NCBI. database. EbPKCβII. have. [PKCβ the. same. sequences in conserved regions (C1, C2, C3, and. read.. C4 domain), but not in the C-terminal called the V5 domain. C1 domains were highly conserved. Ⅲ. Results. DAG/PMA binding sites with a characteristic HX₁. 1. Cloning and sequence analyses of EbPKC βI and βII In order to identify the partial sequences of EbPKCβ, databases of other PKCs were obtained using NCBI sequence data. These sequences were used to design degenerated primers. The initial partial. sequences. were. obtained. through. PCR. amplification of inshore hagfish cDNA, including the brain, tentacle, gill, root of the tongue, spinal cord, heart, liver, gut, muscle, skin, and mucous. ₂CX₂CXnCX₂CX₄HX₂CX₇C. motif. (H,. histidine; C, cysteine; X, any other amino acid; n is 13). C2 domains contained a calcium binding loop, which has several highly conserved Asp residues, and a RACK (receptor for activated C-kinase) binding site. It was confirmed that both EbPKCβI and βII had these highly conserved motifs. Kinase domains include an ATP-binding site (GXGXXG; G is glycine and X is any other amino acid), invariant Lys, and gatekeeper residue. cPKCs and nPKCs. - 1689 -.
(12) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE have Met as a gatekeeper residue, while aPKCs use. nPKCs,. Ile. It was confirmed that EbPKCβI and βII have. species, βI was more closed compared to the βI of. these residues as well. In the case of V5 domains,. another species but not in the case of fish (DrPKC. which are about 50 residues of C-terminus, EbPKC. βI and EbPKCβI).. βI and βII differ significantly. Thus, βI and βII were determined by referencing previous studies [Fig. 3].. and. aPKCs. were. classified.. In. other. 3. Tissue distribution of EbPKCβI and βII by qRT-PCR analysis The distributions of EbPKCβI and βII transcripts. 2. Phylogenetic tree of EbPKCβI and βII In. order. to. determine. the. in different organs were examined by RT-PCR. The. evolutionary. relationship of EbPKCβI and βII with other families of the PKC, a phylogenetic tree was constructed. Phylogenetic analysis was performed with the amino acid sequences of human PKCs and the PKCs of other species obtained from GenBank. results of qPCR indicated that EbPKCβI and βII were expressed in different organs, including the brain, tentacle, gill, root of tongue, spinal cord, heart, liver, gut, muscle, skin, and mucous gland. The expression pattern of EbPKCβI was found at its highest levels in the root of the tongue and spinal cord [Fig. 5A]. The expression pattern of. using neighbor-joining methods [Fig. 4]. Based on. EbPKCβII was found at high levels in the gill,. a. liver, and gut [Fig. 5B].. comprehensive. phylogenetic. analysis,. cPKCs,. [Fig. 4] Phylogenetic relationship of EbPKCβI and βII with other PKC subtypes. In this neighbor-joining phylogram, all individuals are represented and the branches are based on the number of inferred substitutions, as indicated by the bar. The square indicates EbPKCβI and EbPKCβII.. - 1690 -.
(13) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri) ((B) 18 a. EbPKCbetaI. Relative mRNA expression target gene/18s-rRNA. a. b. 14. c. cdef. defg 4. c. 2. efg fg. g. 0. ro. c. bcde. 6. spi nal cgoir lld. m u cou s gml u ski ansgcdlue n t. o t o ft tenhebto tac rangu le in e. c. c. bcd. bcde. 8. t. live r. hea rt. spi nal c. c. bc. 10. c c. b. 12. hea r. c. EbPKCbetaII. 16. live r. ogrildl ro. 158 156 154 152 150 148 146 144 142 140 20 18 16 14 12 10 8 6 4 2 0. mu cou s gml u ski anscgdlu n et. Relative mRNA expression target gene/18s-rRNA. (A). ot o f tetnhe tbo tac rnagi u le n e. [Fig. 5] Tissue-specific distribution of EbPKCβI and βII Quantitative real-time PCR of EbPKCβI and EbPKCβII in various tissues. Mean of mRNA levels in E. burgeri tissues were analyzed by real-time PCR, and 2−ΔΔCt levels were calculated relative to the tissue with the lowest expression (PKCβI from gut tissue and PKCβII from muscle tissue) set to 1 and normalized against 18s-rRNA expression. Each experiment was done in triplicate.. ((B). (A). [Fig. 6] SDS-PAGE and Western blot analysis of EbPKCβI and βII. 10% SDS-PAGE gel and coomassie R-250 blue was used to perform SDS-PAGE. Anti-His antibody was used for Western blotting. Predicted recombinant EbPKCβs Molecular weight is approximately 92 kDa, as they include Trx-tag, S-tag, His-tag, and other amino acids. M, standard size marker; N, cell lysate from IPTG-not induced EbPKCβ-expressing E. coli strain BL21 (DE3); I, cell lysate from 1 mM IPTG-induced EbPKCβ-expressing E. coli strain BL21 (DE3).. 4. SDS-PAGE and western blot. βII, transformed cells were grown in LB broth (5. In order to select recombinants of EbPKCβI and. ml) containing ampicillin (100 µg/ml) and had IPTG added to a final concentration of 1 mM. The. - 1691 -.
(14) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE sample. was checked with induction of target. activity assay using substrate which could be. proteins by SDS-PAGE (10% running gel, 5%. phosphorylated. by. PKC.. stacking gel) and Western blotting was performed. identified. they. can. [Fig. 6A, 6B].. substrate [Fig. 8].. Although the size of EbPKCβI and βII is approximately 76 kDa in both cases, the target size. that. Consequently, indeed. we. phosphorylate. (A) 1. on SDS-PAGE is about 92 kDa, because pET32b. 2. 3. 4. 5. vector expresses other proteins, including Trx tag, S tag, and His tag. Recombinant EbPKCβI and βII were purified by affinity column with nickel resin at. 4°C.. SDS-PAGE. was. onducted. with. 10%. acrylamide gel [Fig. 7A, 7B]. Purified samples were dialyzed in order to check the EbPKCβ ’s. (B) 1. activities.. 2. 3. 4. 5. (A). (B). [Fig. 7] SDS-PAGE of EbPKCβI and βII purified by affinity chromatography. 10% SDS-PAGE gel and coomassie R-250 blue was used to perform SDS-PAGE. Predicted recombinant EbPKCβs Molecular weight is approximately 92 kDa. M, standard size marker; -10, sample obtained for 10 minutes after passing through an elution buffer; 1~10, sample obtained for the next minute; +10, sample obtained during the last 10 minutes.. [Fig. 8] Qualitative analysis of EbPKCβI and EbPKCβII. Substrate was phosphorylated by purified proteins at 1, 2, 5, and 10 ng/μl, respectively. Lane 1 indicates negative control and lanes 2-5 indicate reactants which contain substrate and diversely diluted purified protein (1, 2, 5, and 10 ng/μl). The upper parts are phosphorylated substrate and the lower parts are non-phosphorylated substrate. The results of quantitative analysis showed that in the case of EbPKCβI, it phosphorylated substrate at 14.52, 22.36, 29.25, and 40.43 pmol/min at 1, 2, 5, and 10 ng/μl, respectively [Fig. 9A]. EbPKCβII phosphorylated substrate at 0.86, 1.27, 10.32, and. 5. Activity assay. 43.87 pmol/mine at 1, 2, 5, and 10 ng/μl,. In order to check whether recombinant EbPKCβI. respectively [Fig. 9B].. and βII proteins have PKC activity, we conducted. - 1692 -.
(15) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri) (A). <Table 2> Sequences used in this study. (B). [Fig. 9] Quantitative analysis of EbPKCβI and EbPKCβII. We checked the amount of phosphorylated substrate treated with EbPKCβI and EbPKCβII 1, 2, 5, and 10 ng/μl. (A) Activity of EbPKCβI, (B) Activity of EbPKCβII.. 4. Discussions In this study, we identified sequences of EbPKC βI and EbPKCβII, which originated from one PKCβ gene, and conducted enzymatic analysis. As a result, EbPKCβI and EbPKCβII were found to. In order to check tissue-specific expression, we. encode 668 and 667 amino acids, respectively.. conducted qPCR. EbPKCβI was highly expressed in. They also showed PKC activity.. the root of the tongue and spinal cord, and EbPKC. - 1693 -.
(16) Hyeon-Kyeong JOㆍJun-Young CHAEㆍHyung-Ho LEE βII was highly expressed in the gill, liver, and gut. In particular, a clearly large amount of mRNA was transcribed in the root of the tongue. In several species, PKCβ is involved in the immune system, such. as. in. immunodeficiency,. immunoreceptor and. the. signaling,. development. and. activation of B cells (Kawakami et al., 2002). Therefore, we predict that the root of tongue can act as not only a predatory organ but also a sensory or immune-related organ through contact with the environment. In the jawed vertebrate, PKC β1 is highly expressed in the brain (Goldberg and Steinberg, 1996; Ohno et al., 1987). However, in hagfish (the jawless vertebrate), PKCβ1 is highly expressed in the spinal cord but not in the brain. It is suggested that the spinal cord of the hagfish is more important than that of a jawed vertebrates in the role of the central nerve system. In vertebrates, PKCβI and PKCβII are regulated by miR-203 and miR-7, respectively. Hagfish has miR-199 gene and miR-203a gene (Heimberg et al., 2010). In this study, EbPKCβI and βII contained miR-199 and miR-203a binding sites in their respective 3’-UTR. Therefore, it is possible that Ebu-miR-199 and Ebu-miR-203a regulate EbPKCβI and EbPKCβII, respectively.. References Baneyx F & Mujacic M(2004). Recombinant protein folding and misfolding in Escherichia coli. Nature biotechnology, 22(11), 1399. Blakey Slatter CA, Kanji H, Coutts CA & Ali DW(2005). Expression of PKC in the developing zebrafish, Danio rerio. Journal of neurobiology, 62(4), 425~438. Bobeszko M, Krzemiński P, Pomorski P, Dygas A & Barańska J(2004). Expression and regulation of phospholipase D isoforms in sphingosine and. phorbol ester-stimulated glioma C6 cells. Biochemical and biophysical research communications, 317(3), 689~696. Cole TR Igumenova TI(2015). Expression and purification of the N-terminal regulatory domain of protein kinase C for biophysical studies. Protein expression and purification, 110, 14~21. Das J(2012). Protein Kinase C: The Drug Target One Must See. Medicinal chemistry, 2(5). Donoghue PC & Purnell MA(2005). Genome duplication, extinction and vertebrate evolution. Trends in ecology & evolution, 20(6), 312~319. Goldberg M & Steinberg SF(1996). Tissue-specific developmental regulation of protein kinase C isoforms. Biochemical pharmacology, 51(8), 1089~1093. Gopal GJ & Kumar A(2013). Strategies for the production of recombinant protein in Escherichia coli. The protein journal, 32(6), 419~425. Heimberg AM, Cowper-Sal R, Sémon M, Donoghue PC & Peterson KJ(2010). microRNAs reveal the interrelationships of hagfish, lampreys, and gnathostomes and the nature of the ancestral vertebrate. Proceedings of the National Academy of Sciences, 107(45), 19379~19383. Kawakami T, Kawakami Y & Kitaura J(2002). Protein kinase Cβ (PKCβ): nomal functions and dieases. The Journal of Biochemistry, 132(5), 677~682. Newton AC(1995). Protein kinase C: structure, function, and regulation. Journal of Biological Chemistry, 270(48), 28495~28498. Ohno S, Kawasaki H, Imajoh S, Suzuki K, Inagaki M, Yokokura H, ... & Hidaka H(1987). Tissue-specific expression of three distinct types of rabbit protein kinase C. Nature, 325(6100), 161. Ohno S, Wolf U & Atkin NB(1968). Evolution from fish to mammals by gene duplication. Hereditas, 59(1), 169~187. Ono Y, FuJII TOMOKO, Igarashi K, Kuno T, Tanaka C, Kikkawa U & Nishizuka Y(1989). Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence. Proceedings of the National Academy of Sciences, 86(13), 4868~4871. Schreiber KL, Paquet L, Allen BG & Rindt H(2001).. - 1694 -.
(17) Molecular Cloning, Expression, and Enzymatic Analysis of Protein kinase C βI and βII from Inshore hagfish (Eptatretus burgeri) Protein kinase C isoform expression and activity in the mouse heart. American Journal of Physiology-Heart and Circulatory Physiology, 281(5), H2062-H2071. Steinberg SF(2008). Structural basis of protein kinase C isoform function. Physiological reviews, 88(4), 1341~1378. Wightman PD & Raetz CR(1984). The activation of. protein kinase C by biologically active lipid moieties of lipopolysaccharide. Journal of Biological Chemistry, 259(16), 10048~10052.. •Received : 10 August, 2018 •Revised : 31 August, 2018 •Accepted : 17 September, 2018. - 1695 -.
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인포그래픽이란 어휘 자체는 정보를 나타내는 Information과 그래픽(Graphic)의 합성어이다.. 정보를 구체적이고 실용적이고
통합과학 문학 간호의기초
We determined the nucleotide sequences of the mitochondrial DNA (mtDNA) control region using cloning and sequencing, and obtained the complete sequence from the cattle bones
효율적인 태양광 발전을 위해 필요한 조건들을 생 각해보자..
